Figures

Abstract

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral
infections. HIV-infected individuals develop CTL responses against epitopes
derived from viral proteins, but also against cryptic epitopes encoded by viral
alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape
from CTLs targeting one such cryptic epitope, Q9VF, encoded by an
HIVgag ARF and presented by HLA-B*07. Using PBMCs of
HIV-infected patients, we first cloned and sequenced proviral DNA encoding for
Q9VF. We identified several polymorphisms with a minority of proviruses encoding
at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine
(Q9VF/5N). We compared the prevalence of each variant in PBMCs of
HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were
significantly less represented in HLA-B*07+ than in HLA-B*07-
patients, suggesting that Q9FV/5D encoding viruses might be under selective
pressure in HLA-B*07+ individuals. We thus analyzed ex
vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around
16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF
epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D
or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of
the same CTLs on the two peptides. We then dissected the cellular mechanisms
involved in the presentation of Q9VF variants. As expected, cells infected with
HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In
contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by
Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions
and MS/MS analysis, we demonstrate that the 5N variation introduces a strong
proteasomal cleavage site within the epitope, leading to a dramatic reduction of
Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL
surveillance by introducing mutations leading to HIV ARF-epitope destruction by
proteasomes.

Author Summary

In addition to the classical open reading frames encoding for the well
characterized HIV proteins, HIV exhibits a vast number of alternative reading
frames that have the potential to encode proteins or polypeptides. We have
previously shown that such reading frames within gag, pol and
env genes express T cell epitopes. In the present work, we
further characterized the role of T-cell responses targeting the
gag-overlapping reading frame in the selection of HIV
variants in vivo. We demonstrate that under CD8+ T cell
immune pressure, HIV escapes by introducing mutation that affects T-cell
recognition of HIV-infected cells. We characterized the mechanism of CTL-escape
and demonstrate that HIV manipulates antigen processing and presentation. Our
results highlight the importance of CTL targeting these alternative reading
frame-encoded antigens in the control of HIV replication.

Funding: This work was supported by the Agence Nationale de Recherche sur le SIDA et les
hepatitis virales (ANRS), Sidaction, the Deutsche Forschungsgemeinschaft (DFG),
the University Pierre et Maris Curie (UPMC), INSERM, CNRS and the Institut
Pasteur. We also thank the NIH AIDS Research and Reference Reagent Program for
providing drugs and compounds. R. Bouziat is a fellow of the ANRS. S. Cardinaud
is supported by ANRS and Sidaction. A. Urrutia is supported by Sidaction. The
funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Multiple lines of evidence suggest that CD8+ cytotoxic T lymphocytes (CTLs) play
a critical role in controlling HIV-1 replication. During acute infection, expansion
of HIV-specific CD8+ T cells (HS-CTL), before appearance of neutralizing
antibodies, is associated with decreased viremia [1] and most likely determines the
viral set point during chronic infection [2], [3]. Resistance to disease progression
correlates with the detection of Gag-specific CTLs and with the presence of
particular HLA alleles, such as HLA-B*57 and –B*27 [4], [5]. HIV rapidly
mutates to evade virus-specific CD8+ T lymphocyte responses, underlying the
selection pressure exerted by CTLs [2], [6], [7]–[11]. In large part due to its
error prone reverse transcriptase activity, HIV possesses a unique capacity to
mutate and evade CTL responses. During acute and chronic HIV infection, CTL escape
mutations have been well documented [9], [12], [13]. In most cases, these
mutations are intra-epitopic and affect HLA binding and/or alter TCR interactions
leading to loss of CTL activation or more subtle effects [14]. However, interference with
antigen processing may also lead to a reduced generation of precursor peptides and
consequently peptide/MHC-I complex formation and T cell activation. This could occur
at any stage of the processing pathway. Mutations in epitope-flanking regions might
affect proteasomal processing or N-terminal trimming leading to escape from CTL
recognition [15]–[20].

CTLs recognize peptides originating from proteasomal processing of viral proteins or
truncated misfolded viral polypeptides, also called DRiPS (for defective ribosomal
products) [21]–[23]. These viral polypeptides are classically derived from
the fifteen HIV-1 viral proteins encoded by the nine primary open reading frames
[24].
However CTLs also target peptides translated from alternative reading frames or ARFs
(also called cryptic epitopes). ARF-derived peptides (ARFPs) result from a
differential usage of the three-letter codon alphabet during protein synthesis. How
this change of reading frame occurs remains elusive but various mechanisms have been
proposed. Ribosomes can initiate translation at an internal initiation codon (Met or
Cys), change reading frame by shifting, or translate alternatively spliced mRNA.
Nonetheless, ARF polypeptides are processed in cells and thus constitute an
important source of cryptic epitopes for MHC-I presentation [25]. CTL responses directed against
these cryptic epitopes have been detected in autoimmune disease [26], in tumors
[27], [28] but also in
several infectious diseases, including influenza virus [29], murine AIDS [30], SIV [31] and importantly
HIV infections [32]–[35].

We previously described six ARFPs presented by HLA-B*0702 overlapping the
alternative reading frames of HIV-1 gag, pol or
env genes [32]. CTL responses specific for these ARF-derived peptides
were detected in the blood of HIV+ patients. In addition, HIV-infected cells
were recognized by CTLs specific for the gag-overlapping ARF
epitope (so called Q9VF/5D epitope). Importantly, we showed that the introduction of
a stop codon within gag-ARF abrogated Q9VF/5D epitope generation
and Q9VF/5D–specific CTL activation [32]. Recent studies further
highlighted the in vivo relevance of ARFP-specific CTL responses
[33], [34], [36]. In two
independent cohorts studies, Bansal et al. and Berger et
al. investigated the association between specific HLA alleles and HIV
sequence polymorphisms within ARFs. This “HLA class I footprint
approach” allowed the prediction of numerous ARFPs within the HIV-1 genome,
both from sense and antisense transcripts. On a restricted number of ARFPs, they
also demonstrated that these cryptic epitopes induced CTL responses during natural
infection that might contribute to viral control in vivo[33], [34].

In the present work, we bring to light a novel mechanism of CTL escape altering the
processing and presentation of the Q9VF epitope encoded by the
gag-overlapping ARF. In PBMCs of HLA-B*07+ and
HLA-B*07- HIV-infected individuals, we first compared the prevalence of
QPRSNTHVF (Q9VF/5N) and QPRSDTHVF (Q9VF/5D) variants of the
gag-ARFP. To this end, we PCR amplified and sequenced twenty HIV
proviral genomes per individuals. We noticed that the proportion of proviruses
encoding Q9VF/5D was significantly lower in HLA-B*07+ than in HLA-B*07-
patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure
in HLA-B*07+ individuals. In HLA-B*07+ and HLA-B*07- patients,
we analyzed ex vivo CTL responses directed against Q9VF/5D and
Q9VF/5N and we dissected the immunogenicity of Q9VF variants. We observed that cells
infected with HIV-1 strains encoding Q9VF/5N were neither recognized by Q9VF/5N- nor
Q9VF/5D-specific CTLs. We demonstrate that this single amino acid (AA) variation is
responsible for the lack of CD8+ T cell recognition. We show that HIV can
escape CTL surveillance by introducing mutations leading to epitope destruction by
proteasomes.

Results

Q9VF was originally predicted from the sequence of the consensus
HIVHxB2 (HIVLAI) isolate [32]. HIVLAI bears
an asparagine (N) to aspartic acid (D) substitution at position 5 (Q9VF/5D)
representing less than 5% of HIV-1 clade B strains retrieved from
Genbank. We decided to extend these observations by sequencing HIV proviral
sequences isolated from 10 HLA-B*07+ and 10 HLA-B*07- patients.
HLA-typing, virological and clinical characteristics of these patients are
presented in Table 1. Both
groups were age-matched and did not present any significant differences in terms
of CD4 counts, viral loads or treatments (not shown). From the PBMCs of each
patient, we cloned and sequenced at least 20 HIV-proviral sequences encompassing
the gag-ARF DNA region (Figure 1A and Supplementary Figure S1).
The isolated HIV sequences encoded either Q9VF/5N (present in 16 out of 20
patients, representing 62% of all isolates), Q9VF/5N variants (exhibiting
within the epitope an additional AA difference from the consensus sequence, 9
out of 20 patients, 14% of all isolates) or Q9VF/5D (7 out of 20
patients, 15% of all isolates) and Q9VF/5D variants (2 out of 20
patients, 1% of all isolates) (Table 2). Between Q9VF/5N and
Q9VF/5N-variants, Q9VF/5N was the major variant representing 80% of
proviral sequences in this group. Q9VF/5D was the major sequence representing
94% of proviral sequences among Q9VF/5D and Q9VF/5D-variants. Note that
these mutations did not impact the translation of classical gag
ORF (Supplementary Figure S1 and not shown). In contrast, HIV
proviruses harboring a STOP codon prior to Q9VF (8% of all isolates) that
most likely abolishes Q9VF translation were also identified (Figure 1A). HIV proviral
sequences encoding Q9VF/5N and Q9VF/5N-variants were predominant in both
HLA-B*07+ and HLA-B*07- patients. Q9VF/5D or Q9VF/5D-variant HIV
proviral sequences could be retrieved in two out of the ten HLA-B*07+
patients and in six out of the ten HLA-B*07- donors. Taking into
consideration the diversity of HIV sequences per donor with regard to their
HLA-B7 status, we observe a significant lower proportion of Q9VF/5D+ HIV
strains in HLA-B*07+ than in HLA-B*07- donors (p<0.04, mean
value 3% vs 29% of proviral sequences in HLA-B*07+ and
HLA-B*07- donors, respectively, Figure 2B). Altogether, these results suggested that
Q9VF/5D-encoding HIV strains might be under negative selective pressure in
HLA-B*07+ donors. We thus analyzed CTL responses directed against
Q9VF/5D and Q9VF/5N epitopes in PBMCs of patients including the 10
HLA-B*07+ patients used for the analysis of HIV proviral sequences.

(A) Analysis of Q9VF proviral sequences in HIV-infected
donors. Using PBMCs, proviral DNA of 20 HIV+ individuals were
extracted and the region corresponding to gag-ARF
PCR-amplified and cloned. Twenty clones per donor were sequenced.
Results are presented as percentage of provirus encoding for Q9VF/5D and
5D variants exhibiting within the epitope an additional AA difference
from the consensus sequence, Q9VF/5N and 5N variants, and sequence
harboring a stop codon prior the epitope (no epitope). Pies on the right
represent percentage of provirus combined for all isolates. Top and
bottom panels, results for HLA-B*07+ and HLA-B*07- donors,
respectively. (B) Percentage of provirus encoding Q9VF/5D
or 5D variants within HLA-B*07+ and HLA-B*07- patients.
Each dot represents percentage within the PBMCs of one donor. In
HLA-B*07+ patients, variants with 5D are under-represented
(P<0.04). (C) Immunogenicity of Q9FV peptide variants.
PBMCs of HIV-infected HLA-B*07+ donors were loaded with
peptides and T cell activation monitored by IFNγ-ELISot. PBMCs were
incubated with HLA-B*07-restricted epitopes: Q9VF/5D, Q9VF/5N, a
pool of 3 immunodominant HIV-1 Gag epitopes (SPRTLNAWV, TPQDLNTML,
YPLASLRSLF), a CMV-derived epitope (pp65 TPRVTGGGAM) or an HCV-derived
epitope as negative control (GPRLGVRAT). Out of 31 HLA-B*07+
patients 5 reacted to Q9VF/5D and Q9VF/5N. Results for the 5 Q9VF
reacting patients (Q9VF CTL +, full symbols) and 5 representative
Q9VF non-reacting patients (Q9VF CTL-, open symbols) are shown. Data are
means of triplicates. Dotted line indicates threshold of significant
positive responses.

(A) T1-B7 cells were infected with HIVLAI and
HIVNL-AD8 expressing Q9VF/5D and Q9VF/5N, respectively.
Two days p.i., the percentage of HIV-infected cells was monitored by
intracellular p24 staining and flow cytometry: 50 and 47% of the
cells were infected with HIVLAI and HIVNL-AD8,
respectively. In an IFNγ-ELISpot assay, infected cells were then
used to activate CTL lines specific for Q9VF/5D, Q9VF/5N or an
HLA-B*07-restricted HIV-1 Nef epitope (FPVTPQVPLR, F10LR) used as
control. For each peptide, specific CTL lines were generated in three
different HLA-B*0702 transgenic mice and used in two independent
experiments. One representative experiment with one CTL line is shown
(mean values of triplicates ±SD). T1-B7 cells loaded with the
cognate peptide were used as positive controls. (B) 5N
substitution does not affect HIV replication. T1-B7 cells (left panel)
and CD4+ activated T cells (right panel) were infected (at 100 and
1 ng/ml respectively) with HIVLAI and
HIVLAI-5D>5N. HIVLAI-5D>5N expressing
Q9VF/5N was engineered by PCR mutagenesis of the HIVLAI
strain. Whatever the viral input (1, 10 or 100 ng/ml), 5N substitution
did not alter the replication capacity of HIVLAI-5D>5N.
T1-B7 cell infection (left panel) was monitored using GFP expression
(upon trans-activation of LTR-GFP). Data are representative of at least
five independent experiments using various viral inputs. CD4+ T
cells infection was monitored using p24-Elisa (right panel) and
correspond to the mean values (±SD) of two infections using
activated CD4+ T cells from two donors and are representative of
two independent experiments (using various viral input). NI: not
infected. (C) 5N substitution is sufficient to abrogate CTL
recognition of HIV-infected cells. As in (A) using T1-B7 cells infected
with HIVLAI, HIVNL-AD8 and
HIVLAI-5D>5N. Infection rates were around 30%
of p24+ cells.

PBMCs from 31 HLA-B*07+ patients were loaded with various peptides and
submitted to IFNγ-ELISpot (Figure 1C and not shown). Incubations with peptides corresponding to
well-characterized HLA-B*0702-restricted immunodominant epitopes from HIV-1
Gag classical ORF (SPRTLNAWV, TPQDLNTML, YPLASLRSLF) induced a significant
IFNγ-release, demonstrating that in the course of natural infection the
donors mounted CTL responses to HIV-1 antigens. Five out of the 31
HLA-B*07+ donors showed a low but significant activation with Q9VF/5D
and Q9VF/5N peptides (Figure
1C). Note that donors reacted to both peptides or reacted to none and
that the frequencies of CTL responding to Q9VF/5D and Q9VF/5N peptides were in
the same order of magnitude (from 150 to 300 CTL per million of PBMCs),
suggesting that the reactivity to one or the other peptide might be due to cross
reactivity. We previously demonstrated that CTL lines raised against Q9VF/5N
were indeed cross-reactive on Q9VF/5D and vice versa ([32] and
Supplementary Figure S2).

Viruses encoding Q9VF/5D were not isolated from PBMCs of the five Q9VF responders
(Figure 1), with the
exception of patients P1 that harbored proviruses encoding a Q9VF/5D variant
(QPRGDTHVF, representing 16% of sequences in this
donor). These data prompt us to study the immunogenicity of the Q9VF/5N and
Q9VF/5D epitope variants.

We asked whether the Q9VF/5N epitope was processed and presented to HS-CTLs by
HIV-infected cells. HLA-B*0702+ cells were infected with
HIVLAI and HIVNL-AD8 strains encoding Q9VF/5D or
Q9VF/5N respectively. Five days post-infection (pi), 50 and 47% of the
cells were productively infected by HIVLAI and HIVNL-AD8
respectively (as monitored by intracellular Gag-p24 FACS-staining (not shown)).
Infected cells were then co-cultured with HIV-specific CTL lines and T cell
activation measured using IFNγ-ELISpot assays (Figure 2). HLA-transgenic mice offer a rapid
and convenient model to identify human T cell epitopes [24] and to generate CTL
lines specific for peptides of unknown immunogenicity in humans, such as
Q9VF/5N. For this reason, Q9VF/5D- and Q9VF/5N-specific CTL lines were generated
by peptide immunization of HLA-B*0702+ transgenic mice and in
vitro restimulations [32], [37]. As expected, Q9VF/5D-
and Q9VF/5N- specific CTLs secreted high levels of IFNγ in response to
Q9VF/5D and Q9VF/5N peptide loaded cells respectively (Figure 2A). Note that Q9VF/5D- and
Q9VF/5N-specific CTL lines displayed similar capacity to recognize
peptide-loaded cells (Supplementary Figure S2), suggesting that the Q9VF/5N
variant affects neither MHC nor TCR binding of the peptide. As we previously
reported [32], HIVLAI-infected cells induced a robust
activation of Q9VF/5D-specific CTLs. Due to their capacity to cross-react on
Q9VF/5D peptide (Supplementary Figure S2 and [32]), Q9VF/5N-specific CTLs
were also stimulated by HIVLAI-infected cells, thus demonstrating
that these CTL lines are fully competent in recognizing HIV-infected cells. In
contrast, Q9VF/5D- and Q9VF/5N-specific CTLs were not activated upon co-culture
with HIVNL-AD8-infected cells (Figure 2A). This is not due to the incapacity
of HIVNL-AD8-infected cells to activate HS-CTLs since CTL clones
specific for an HLA-B*0702-restricted HIV-1 Nef epitope (F10LR), raised as a
control in these experiments, were activated upon co-culture with
HIVLAI- and HIVNL-AD8-infected cells.

To extend these observations to other HIV-1 isolates, HLA-B*0702+ cells
were also infected with HIVMN that encodes for Q9VF/5N and used as
target cells to activate Q9VF/5D- and Q9VF/5N-specific CTLs (Supplementary Figure S3).
HIVNL-AD8- and HIVMN-infected cells did not induce
Q9VF/5D- nor Q9VF/5N-specific CTL activation. Overall, these results suggested
that HIV-infected cells did not present the Q9VF/5N peptide.

Epitope flanking regions have a direct impact on antigen processing and
presentation [38]. Thereafter, to exclude the possibility that HIV
sequence variations outside the Q9VF/5N peptide might be responsible for the
lack of presentation, we introduced in HIVLAI a D to N mutation
within the Q9VF epitope (so called HIVLAI-5D>5N). This mutation
did not affect the primary open reading frame of Gag (Supplementary Figure S1)
and did not alter viral replication in T cell lines or primary CD4+ T cells
(Figure 2B). However,
cells infected with HIVLAI-5D>5N could not activate Q9VF/5D- nor
Q9VF/5N-specific CTLs (Figure
2C). Thereafter, this single amino acid substitution was sufficient
to abrogate CTL recognition, thus indicating that this asparagine alters Q9VF
MHC-I presentation. We then sought to dissect the mechanism responsible for the
lack of Q9VF/5N MHC-I presentation.

Q9VF/5N binds TAP pumps and HLA-B*0702 molecules

The capacity of antigenic peptides to bind to a given HLA allele is determined by
the so-called anchor residues [39]. Mutating an anchor residue abrogates peptide
HLA-binding and subsequent T cell activation, a strategy often used by viruses
to escape viral-specific T cell responses. The anchor residues of HLA-B*0702
reside at position 2 and 9 of the peptide-ligands. Thereafter, the D to N
substitution at position 5 was not predicted to influence Q9VF peptide binding
to HLA-B*0702 [40]. However, besides anchor residues, auxiliary residues
might affect peptide binding, we thus compared the capacity of Q9VF/5D and
Q9VF/5N peptides to bind HLA-B*0702. To this end, T2-HLA-B*0702 cells
were loaded O/N with Q9VF/5D or Q9VF/5N peptides and binding to HLA-B*0702
molecules at the cell surface monitored by FACS (Figure 3A, left panel). Q9VF/5D and Q9VF/5N
peptides exhibited similar capacities to bind HLA-B*0702 with a relative
affinity (RA, based on the reference peptide) of 2.6 and 1.5 respectively (Figure 3A, left panel). To
further characterize the impact of the 5D to 5N substitution on peptide-MHC
interactions, we compared the capacity of the peptides to stabilize
HLA-B*0702 molecules at the cell surface of T2-HLA-B*0702 (Figure 3A, right panel). To
this end, T2-HLA-B*0702 were cultured O/N at 26°C to allow surface
expression of peptide-receptive MHC molecules, loaded with a high concentration
of peptides, shifted to 37°C and the stability of HLA-B*0702-peptide
complexes monitored by FACS at various time points. An exponential regression of
HLA-B*0702 mean fluorescence intensity (MFI) vs. time reveals that the
stability (t1/2) of HLA-B*0702 pulsed with an irrelevant peptide
(S9L) is 22 min while binding of Q9VF/5D and Q9VF/5N peptides prolongs the
t1/2 to 211 and 641 min respectively (Figure 3A, right panel). Thereafter, Q9VF/5D
and Q9VF/5N peptides are very good HLA-B*0702-binders and 5D to 5N
substitution tends to prolong surface expression of HLA-B*0702.

(A) Q9VF/5N and Q9VF/5D peptides exhibit similar affinities
for HLA-B*0702. (Left panel) Q9VF/5D, Q9VF/5N and
their natural EGF Nt-extended precursors were loaded O/N at RT on T2-B7
cells. An HLA-B*07-restricted CMV-derived reference epitope (pp65
RPHERNGFTV, R10TV) and an HLA-A*02-restricted HIV-1-derived epitope
(p17 SLYNTVATL, SL9) were also used as positive and negative control,
respectively. HLA-B*0702 binding was monitored using ME-1 antibody
and flow cytometry. Based on the reference peptide R10TV, a relative
affinity (RA) was calculated. Data are representative of three different
experiments (mean values of triplicates ±SD). (Right
panel) T2-B7 were cultured O/N at 26°C to increase
peptide-receptive cell surface molecules, pulsed with the indicated
peptides for 2 h in presence of β2-microglobulin and BFA to stop
delivery of newly synthesized MHC-I molecules. Cells were then shifted
to 37°C for 1 h, washed to remove unbound peptides and incubated at
37°C in presence of BFA (0.5 µg/ml) which is considered as
time “zero”. At the indicated time points, samples were
removed to 0°C, stained on ice using ME.1 Ab and analyzed by FACS.
Data are mean values of two independent experiments. The capacity of
each peptide to stabilize HLA-B*0702
(t1/2) was compared using exponential
regression. T1/2 of HLA-B*0702 pulsed with the irrelevant
peptide (S9L) was 22 min while binding of Q9VF/5D and Q9VF/5N peptides
prolonged the t1/2 to 211 and 641 min respectively.
T1/2 of CMV (pp65 TPRVTGGGAM, T10AM) and Gag (p24
TPQDLNTML, T9ML) peptides used as positive were 552 and 124 min
respectively. (B) Human TAP transporter binding assay.
Microsomes from insect cells expressing human TAPs were incubated with
the labeled reference reporter peptide (RRYNASTEL, R9L) then loaded with
serial dilutions of unlabeled reference peptide or tested peptides with
or without EGF Nt-extension. TAP affinities were determined as the
concentrations required to inhibit 50% of reporter peptide
binding (IC50) and data are presented as 1/IC50
ratios: the highest the ratio, the stronger the affinity. Results are
mean values (±SD) from three independent experiments.
(C) Q9VF/5D epitope generation is dependent on
proteasomal processing. T1-B7 cells were infected with HIVLAI
(as in Figure 1),
monitored for HIV infection by flow cytometry, treated or not (unTx)
with epoxomicin (6 h at 37°C). To remove residual MHC-peptide
complexes, cells were then treated with a citrate-phosphate buffer,
washed and used as targets to activate Q9VF/5D-specific CTLs in
IFNγ-ELISpot assay (8h). Note that epoxomycin inhibition affected
neither MHC-density (as monitored by FACS, not shown) nor the capacity
of treated cells to present exogenous peptide (0.1 µg/ml) (right
panel). Results are mean values (±SD) of triplicates and
representative of three different Q9VF/5D CTL clones. Mock, non infected
cells (left panel) or loaded with the irrelevant HCV peptide (right
panel).

Precursor peptides are transported by the TAP pumps (transporter associated with
antigen processing) from the cytosol into the endoplasmic reticulum (ER), and
then loaded on nascent MHC-I molecules [41]. N-terminally extended
peptide precursors are also transported and further trimmed in the ER by the
endoplasmic reticulum aminopeptidase ERAAP and bound to MHC-I molecules [42], [43]. We asked
whether the absence of Q9VF/5N peptide presentation by HLA-B*0702 within
infected cells might be the result of inefficient ER-translocation of the
Q9VF/5N epitope and/or Q9VF/5N-peptide precursors by TAP. Hence, we used a
TAP-binding assay [44] to evaluate the affinities of Q9VF/5D and Q9VF/5N and
their precursors with TAP. Q9VF/5D and Q9VF/5N exhibited a poor affinity for TAP
(Figure 3B), most likely
due to the presence of a proline at position 2 that negatively impacts on
TAP-mediated peptide transport [44]. In contrast, their
N-terminally extended peptide precursors EGF-Q9VF/5D and EGF-Q9VF/5N showed at
least a two-log increased efficiency to compete for TAP with an equal
1/IC50 of 0.15. Whatever the precursor, Q9VF/5D and Q9VF/5N
containing peptides did not show differences in their capacity to bind human TAP
molecules.

Overall, these data demonstrated that the D to N substitution within Q9VF does
not impact on TAP transport and HLA binding. In contrast, the 5N substitution
might prolong epitope presentation on the cell surface.

Q9VF/5D epitope generation is dependent on proteasomal cleavages

The proteasomes, that are the major catalytic enzymes involved in antigen
processing, generate the carboxyl termini of most MHC-bound peptides [38], [45]. We thus
asked whether the generation of Q9VF/5D was dependent on proteasomal processing.
To this end, HLA-B*0702+ cells were infected with HIVLAI.
Five days pi, infected cells were incubated with a potent and selective
proteasome inhibitor, epoxomicin [46], treated with a citrate-phosphate buffer to remove
residual MHC-peptide complexes, washed and cultured with Q9VF/5D-specific CTLs
as previously described. Epoxomicin treatment abolished the capacity of
HIVLAI-infected cells to activate Q9VF/5D-specific CTLs, as
measured in IFNγ-ELISpot (Figure 3C, left panel). Note that epoxomycin inhibition affected
neither MHC-density (as monitored by FACS, not shown) nor the capacity of
treated cells to present exogenous peptide (at 0.1 µg/ml) (Figure 3C, right panel).
Thereafter, these results demonstrated that the generation of Q9VF epitope
depends on proteasomal processing.

5N introduces an aberrant proteasomal cleavage site within Q9VF
epitope

Proteasomes might also destroy CTL epitopes by generating aberrant cleavages
within the epitope [47] or in epitope-flanking regions [19], [48]. We thus asked whether
aberrant proteasomal cleavages might be responsible for the lack of Q9VF/5N
presentation.

The proteasome is a large multicatalytic protease composed of standard and
inducible subunits that replace the standard subunits upon exposure to IFNγ
and form the so-called “immunoproteasomes” (IP). IP is found in most
cell types after IFNγ-exposure, but is constitutive in APCs and is induced
in HIV-infected T cells [49]. Standard (SP) and IP proteasomes display discrete
differences in their capacity to cleave a given peptide substrate [50]. We submitted
the full-length polypeptides from the gag-overlapping ARF to IP
processing. 27mer peptides encompassing Q9VF/5D or Q9VF/5N peptides were
synthesized and incubated with IP purified from T2.27 cells [51]. After
1 h incubation, the digestions were analyzed by mass spectrometry (RP-HPLC SI)
and peptide fragments identified by MS/MS (Figure 4A). IP digestion of Q9VF/5D
encompassing peptide showed the presence of major proteasomal cleavage sites
after amino acids F10, F19, I22 and R24 representing around 80% of total
cleavages. The cleavage at position F19 generated the C-terminal cut of the
N-extended precursors of Q9VF (M1-F19). After 1 h incubation, when comparing the
IP digestion profiles of Q9VF/5D and Q9VF/5N encompassing peptides, we noticed
the presence of a new cleavage site within the Q9VF/5N epitope. This cut at
position N15 was the most prevalent among Q9VF/5N representing up to 28%
of total IP cleavages. These results demonstrated that the D to N substitution
introduces a major cleavage site within the Q9VF/5N epitope. Nonetheless the
C-terminal cut necessary for the generation of Nt-extended Q9VF/5N precursors
was also detected following 1 h of proteasomal digestion.

Figure 4. 5N introduces an aberrant proteasomal cleavage site within the
epitope.

(A) 5N introduces a strong cleavage site within Q9VF
epitope. 27mer synthetic peptides encompassing Q9VF/5D or Q9VF/5N were
submitted to in vitro immunoproteasome (IP) digestion.
Resulting peptide fragments were analyzed by mass-spectrometry.
Proteasome cleavage patterns are presented as C-terminal cleavages to a
specific AA (horizontal axis) of Q9VF/5D (upper panel) and Q9VF/5N
(lower panel) substrates. The percentage of C-terminal cuts at each AA
is indicated. The most frequent fragments at 1 h IP digestion are
depicted. Data represent one of two independent experiments.
(B) The overall production of Q9VF epitope is
drastically reduced by the 5N substitution. Q9VF/5D (upper panel) and
Q9VF/5N (lower panel) encompassing peptides were digested by IP from 0 h
to 18 h. Resulting peptide fragments were analyzed by MS/MS, as in (A).
Proteasome cleavage patterns are presented as the estimated percentage
of peptide fragments corresponding to either the substrate (M1-P27), the
epitope Q9VF (Q11-F19), precursors with a C-terminal cut at F19, peptide
fragments with a cleavage within the epitope most likely abolishing
epitope production (referred to as “Antitopes”), or other
fragments, with the sum of all fragments intensities set as
100%.

Thereafter, we sought to evaluate the amount of cleavage products generated
during Q9VF/5D and Q9VF/5N digestions. To this end, we performed kinetics of IP
digestion where aliquots were regularly collected and submitted to mass
spectrometry analysis as before (Figure 4B). To compare the amounts of cleavage products, we used the
MS fragment intensity as a surrogate marker for quantity since these two
parameters correlate significantly [15]. The variations among the
different fragments generated are presented as the relative intensity of
peptides that exhibit a Q9VF C-terminal cut (epitope or precursors) or peptides
issued from cleavages within the Q9VF epitope (referred to as the antitopes)
(Figure 4B). Kinetics of
digestion of peptides encompassing either Q9VF/5D or Q9VF/5N were identical:
24%, 59% and 96% of both substrates was degraded after 30
min, 1 h and 2 h respectively. At latter time points, both 27mers were
undetectable. In the course of Q9VF/5D substrate digestion, the precursor
(M1-F19) was readily produced starting from 30 min with a peak at 4 h digestion
(representing 20% of digested products). The epitope was detected
starting from 1 h digestion and accumulated reaching 13% of all peptide
fragments at time 18 h. At latter time points, Q9VF/5D epitopes and precursors
represented up to 14% of all peptide fragments detected. An antitope
corresponding to a cleavage at position S14 was also generated but represented
less than 2% of detected fragments at each time point. In contrast,
during Q9VF/5N substrate digestion, the antitopes corresponding to the cleavage
at position N15 were already produced after 30 min of digestion and reached
around 77% of all peptides from 4 to 18 h, further demonstrating that N15
is a major cleavage site within Q9VF/5N. Interestingly, during Q9VF/5N
digestion, the epitope was barely detected even at latter time points (less than
2% of digested products). The precursor M1-F19 accumulated from 30 min to
2 h (8% of digested products) but was undetectable after 4 h, suggesting
that the cleavage at position N15 destroyed this peptide. Overall, the amounts
of Q9VF/5N epitope and precursors produced were markedly reduced as compared to
Q9VF/5D digestion.

Taken together, these results demonstrate that the Q9VF/5D epitope is efficiently
produced by proteasomes and accumulates with time. In contrast, the D to N
substitution introduces a major cleavage site within the epitope leading to the
destruction of the Q9VF/5N epitope and thus the absence of MHC-I binding and
presentation.

Discussion

The three-letter codon alphabet allows protein synthesis in six possible overlapping
reading frames. A vast number of ARFs have the potential to encode proteins or
epitopic peptides (ARFPs). Using an “HLA class I footprint” approach,
Bansal et al and Berger et al recently predicted
the existence of numerous ARFPs within HIV-1 genome [33], [34]. We have previously shown that
ARFP-specific CTLs are induced during natural infection [32]. These CTL responses might
contribute to viral control driving HIV evolution at the population level. ARFPs can
mutate during the first year of infection, suggesting a possible selection of
escapes variants [33], [34]. Such a scenario has been highlighted in the macaque
model of SIV infection [31]. Mamu-B*17+ macaques generate strong CTL
responses against SIV ARF-encoded epitopes leading to ARF mutation affecting epitope
binding to Mamu-B*17 molecules and subsequent SIV replication rebound [31]. In the present
study, we characterized a novel mechanism of ARFP-specific CTL escape resulting from
HIV epitope destruction by the proteasomes. We suggest that ARFP-specific CTLs exert
a selection pressure leading to negative selection of targeted HIV strains. Overall,
our work shows that CTL escape mutations are not limited to epitopes encoded by
classical ORF, highlighting the role of ARFP-specific CTLs in the control of HIV
infection.

We previously identified a panel of epitopes encoded by ARFs within HIV-1
gag, pol and env genes [32]. The
gag-overlapping ARF encoding for the Q9VF epitope presented by
HLA-B*0702 drew our attention due to its polymorphism. In a cross-sectional
cohort study, we report that proviruses encoding the Q9VF/5D epitope (and 5D
variants) are rare and significantly under-represented in PBMCs of
HLA-B*07+ patients, thus suggesting Q9VF/5D-specific CTLs might exert a
negative selection pressure on HIV strains encoding Q9VF/5D variants. In HIV-1
gag ARF, the virus might escape CTL immune pressure by
introducing a 5D to 5N substitution or Stop codons but prior the epitope. We thus
analyzed CTL responses directed against Q9VF/5D and Q9VF/5N epitopes in PBMCs of
patients. Q9VF/5D and Q9VF/5N peptides induced CTL responses in 16% of
HLA-B*07+ individuals tested. Donors reacted to both peptides or reacted to
none. The frequencies of CTLs responding to Q9VF/5D and Q9VF/5N peptides were about
the same magnitude, suggesting that the reactivity to one or the other peptide might
be due to cross reactivity. The frequency and magnitude of Q9VF/5D responses in
HLA-B*07+ patients were rather low as compared to immunodominant
HLA-B*07-restricted responses (Figure 1 and [24]). This might be due to the fact that the patients
included in the study were under retroviral therapy that might affect the expression
of ARF during residual HIV-1 translation (Table 1). Alternatively in our assays, we are
most likely monitoring memory responses to Q9VF/5D that are usually of low
magnitude. This possibility is supported by the observation from Bansal et
al that ARFP encoding sequences mutate during the first year of
infection [33].
Overall, the low representation of Q9VF/5D encoding HIV proviral sequences in PBMCs
of HLA-B*07+ individuals and the low frequency and magnitude of CTL
responses to Q9VF/5D strongly supported our initial hypothesis that 5N substitution
is an escape mutation.

We dissected the immunogenicity of the Q9VF/5N epitope. We showed that cells infected
with HIV-1 strains encoding Q9VF/5N (HIVNL-AD8 and HIVMN) were
not recognized by Q9VF/5N-specific CTLs. In contrast, Q9VF/5N- and Q9VF/5D-specific
CTLs were activated by HIV-1 strains encoding Q9VF/5D (HIVLAI). We
demonstrated that the single AA substitution from 5D to 5N in HIVLAI
sequence is sufficient and required to abrogate CTL recognition of HIV-infected
cells. Thereafter, the acquisition of this 5N mutation by HIV might help the virus
to interfere with Q9VF epitope expression or processing and presentation.

Viruses can interfere with antigen expression to escape CTL lysis [23]. Various
mechanisms have been proposed for the biosynthesis of ARF-derived polypeptides.
Ribosomes can scan through conventional initiation codons [29], initiate translation at an
internal initiation non-AUG-codons (Leu or Cys) [34], [52], change reading frame by
shifting [53], or
translate alternatively spliced mRNA (for review see [25]). We previously described the
presence of a conserved slippery motif (UUUAAAU) upstream of
gag-ARF start codon that may facilitate ribosomal slippage and thus
Q9VF synthesis [32]. Interestingly, a structured region (hairpin) in HIV-1
RNA has been identified downstream of this slippery motif [53]. This highly structured RNA
region might cause ribosomal pausing during gag translation thus
facilitating ribosomal slippery and Q9VF expression. The D to N substitution within
the Q9VF epitope is translated from a codon that is located in the flexible loop of
the RNA hairpin structure [53]. Although it remains to be formally proven, this D to N
substitution most likely does not impact the RNA structure and hence Q9VF
expression.

Viruses also manipulate antigen processing and presentation to escape CTL responses.
Interference with antigen presentation could arise at any stage in the pathway,
including processing by proteasomes, binding of epitope-precursors to TAP,
destruction of these precursors by peptidases in the ER or cytosol and peptide
binding to the MHC-I molecule. HIV-specific CTL responses have been shown repeatedly
to select for intra-epitope mutations that affect HLA-binding or TcR recognition. In
addition, HIV escape mutations outside the epitope (extra-epitope mutations) can
interfere with antigen processing by proteasomes [17]–[19], [47], [54], [55] or by the ER aminopeptidase
ERAAP [16]. To
our knowledge, intra-epitope mutations affecting antigen processing have not been
described thus far. Several studies proposed that intra-epitope variation might
affect processing but did not provide a mechanism [34], [20]. The only evidence that
intra-epitope mutations might affect proteasomal processing of viral antigens comes
from mouse models [47], [56].

We provide several lines of evidence strongly suggesting that the D to N substitution
within the Q9VF epitope impacts neither TcR recognition nor MHC binding: i) Q9VF/5N-
and Q9VF/5D-specific CTLs can be generate upon peptide immunization of
HLA-B*07-transgenic mice and cross-react to the alternate peptide ([32] and
Supplementary Figure
S2); and ii) Q9VF/5N and Q9VF/5D peptides bind HLA-B*0702 (Figure 3A). In addition, we show
that Q9VF/5N and Q9VF/5D peptide and their precursors (elongated on the N-termini)
efficiently bind TAP, thus demonstrating that the D to N substitution does not
affect peptide translocation into the ER. As previously observed with peptides
bearing a proline at position 2 [44], the optimal Q9VF/5N- and Q9VF/5D epitopes had a reduced
capacity to bind TAP as compared to their Nt-extended precursors (Figure 3B), suggesting that in the
ER peptide-trimming is required for proper HLA-B*0702 binding. The ER
aminopeptidase ERAAP provides peptides for many MHC-I molecules but has been also
implicated in the destruction of CTL epitopes [16]. However, ERAAP cannot
process X-P motifs in peptide sequences [42]. Thereafter, though it cannot
be formally excluded, a role of ERAAP in the destruction of Q9VF/5N is very
unlikely. Overall, these data support the concept that the intra-epitope D to N
substitution interferes with proteasomal processing. Using in vitro
proteasomal digestions, we demonstrate that the D to N substitution introduces a
major cleavage site within the Q9VF epitope (at position N15). Note that at 1
h-digestion time point we identify mainly primary cleavage products since less than
50% of the peptide substrates (the 27mer) have been digested (Figure 4A). To further highlight
the potential impact of this N15 cleavage site in the generation of the Q9VF
epitope, we performed kinetics of peptide digestion using IP. We observed that
amounts of Q9VF/5N epitope and precursors produced were markedly reduced as compared
to Q9VF/5D. These results strongly suggest that proteasome cleavages at position N15
destroy the Q9VF/5N epitope and precursors resulting in the lack of MHC-I
presentation and CTL activation. In conclusion, a single amino acid variation within
HIV epitope can result in epitope destruction and absence of HIV-specific CTL
activation.

Mutation in HIV-1 genome can be silent or can differentially impact the fitness of
the virus. Due to the redundancy of the codon alphabet, the 5D to 5N substitution in
Q9VF does not impact the primary gag-ORF and thus viral replication
(Figure 2B). Nevertheless,
considering the multitude of existing ARFs, some mutations within ARF encoding
sequences most likely affect viral fitness and these ARF sequences might be
unavoidably conserved throughout HIV-1 isolates. Thereafter, the great diversity of
ARF epitopes produced during HIV infection offers a vast panel of therapeutic
targets to stimulate CTL responses. It is interesting to note that ARF-specific
CD8+ T cells can performed multiple functions [33], [34] and control viral replication
in vitro, characteristics that correlate with slow disease
progression [57].
In addition, CTLs targeting ARF-derived epitopes can be induced upon vaccination
[58] and tumor
infiltrating CTLs specific for ARFPs have been also identified in various cancers,
including melanoma and breast cancers [25]. Such responses against crytptic epitopes represent a
great potential for future immunotherapeutic strategies.

Materials and Methods

Study population

HIV-1-infected peripheral blood mononuclear cells (PBMCs) were obtained from HCV
(Hepatitis C virus) negative French ALT-ANRS-CO15 cohort patients [59]. The 31
HLA-B*07+ and 10 HLA-B*07- individuals were identified using the
anti-HLA-B*07 antibody ME.1. HLA status was further confirmed by genotyping
using PCR [60]
or using the Luminex xMAP technology [61]. HLA-typing, virological
and clinical characteristics of the ten HLA-B*07+ and ten HLA-B*07-
patients included in the study are presented in Table 1.

Ethics statement

Patient samples were collected according to French Ethical rules. Written
informed consent and approval by institutional review Board at the
Pitié-Salpêtrière Hospital were obtained.

Animals were bred at the Pasteur Institute. The Office of Laboratory Animal Care
at Pasteur Institute reviewed and approved protocols for compliance with the
French and European regulations on Animal Welfare and with Public Health Service
recommendations (Directive 2010/63/EU).

Human CTL assays

PBMCs were isolated by ficoll-centrifugation, pulsed with Q9VF peptides (1
µM, 1 h at 37°C), and submitted to IFNγ-ELISpot assays as
previously described [46]. The HLA-B*0702-restricted peptides used were:
HCV-derived epitope G9AT (GPRLGVRAT), CMV-derived epitope T10AM (pp65
417TPRVTGGGAM426) used as negative and positive
control respectively and a pool of known Gag HIV-1-derived epitopes (p24
16SPRTLNAWV24, p24
48TPQDLNTML56, p2p7p1p6
121YPLASLRSLF130) as control for HIV reactivity [24].
Responses were considered positive when IFNγ production was superior to 50
spots/106 PBMCs and at least threefold higher than background
(measured with the HCV peptide).

HLA-B*07.02-peptide binding and stabilization assays

The capacity of the peptides to bind HLA-B*0702 was determined using a
classical HLA stabilization assays with the TAP-deficient cell line T2
HLA-B*0702+ [37]. Briefly, cells were incubated overnight with 100,
10, 1 and 0.1 µM of peptide in serum-free medium at room temperature.
Cells were then stained with the anti-HLA-B*07 ME.1 antibody and
HLA-B*07 surface expression analyzed by FACS (Becton Dickinson). The
concentration needed to reach 50% of the maximal fluorescence (as defined
with the R10TV peptide (CMV pp65 265RPHERNGFTV274) was
calculated (IC50). The relative affinity (RA) is the IC50
ratio of the tested and R10TV reference peptide (the lower the relative
affinity, the stronger the binding). The HLA-A*02-restricted peptide S9L
(HIV-1 p17 77SLYNTVATL85) was used as negative control. To
monitor the capacity of the peptides to stabilize HLA-B*0702,
T2-HLA-B*0702 were cultured O/N at 26°C and pulsed the last 2 h with
peptide (100 µM) in presence of β2-microglubilin (Sigma, 1
µg/ml) and brefeldin-A (BFA, Sigma, 10 µg/ml). Cells were then
shifted to 37°C for 1 h, washed to remove unbound peptides and incubated at
37°C in presence of BFA (0.5 µg/ml). Samples were removed to 0°C
at the indicated time points. Cells were then stained at 4°C using the ME.1
antibody and analyzed by FACS. Data (HLA-B*0702 expression) are expressed as
MFI vs. time. The capacity of each peptide to stabilize HLA-B*07
(t1/2)is deduced from an exponential regression
(one phase decay) using Prism software. A constrain corresponding to the MFI
value obtained for the irrelevant peptide (S9L) at the latest time point was
applied to the plateaus. T10AM (pp65 417TPRVTGGGAM426) and
T9ML (p24 48TPQDLNTML56) peptides were used as positive
controls.

TAP-binding assay

The capacity of the peptides to bind TAP was measured in a competitive binding
assay as described previously [44]. Briefly, microsomes
were purified from Sf9 insect cells expressing human TAP1–TAP2 complexes,
pulsed with the iodinated reporter peptide R9L (RRYNASTEL) at 300 nM, and loaded
with a dilution of competitor test peptides (0.1 to 1,000 fold molar excess
relative to radioactive reporter peptide). TAP affinities were determined as the
concentrations required to inhibit 50% of reporter peptide binding
(IC50). Results are expressed as 1/IC50 ratios and are
mean values from three independent experiments. The highest the
1/IC50 ratio, the highest the affinity.

In vitro proteasome digestions

Immunoproteasomes were isolated from T2.27mp cells (that stably express all three
immunosubunits) as previously described [51]. Purified proteasomes
were analyzed by SDS-PAGE. The yield was calculated at 90–95%. The
27mer peptides encompassing Q9VF/5D or Q9VF/5N were synthesized using standard
Fmoc method on an Applied Biosystems 433A automated synthesizer. The peptides
were purified by HPLC and analyzed by mass spectrometry. Three nmol of peptides
were digested in vitro using 1 µg of proteasomes (for
0.5, 1, 2, 4, 8 and 18 h) in 100 µl of buffer containing 20 mM Hepes/KOH,
pH 7.8, 2 mM magnesium acetate and 2 mM dithiothreitol. Reactions were stopped
by the addition of trifluoroacetic acid to a final concentration of 0.3%.
The digestions were analyzed, by mass spectrometry (RP-HPLC ESI) and the
products were identified by MS/MS.

Supporting Information

Amino acid and nucleotide sequences of Gag and Gag-ARF. (A)
Nucleotide and corresponding amino acid sequences of Gag (frame 1) and
Gag-ARF (frame 3, bold) are depicted. Nucleotide numbering is according to
HIVHXB2 sequence. ATG start and TGA stop codons of
Gag-ARF are in bold and the Q9VF/5D epitope is
underlined. (B) Nucleotide and amino acid sequences of Gag and
Gag-ARF from HIVLAI, HIVNL-AD8, HIVMN and
HIVLAI-5D>5N strains.

Q9VF/5D and Q9VF/5N CTL cross-reactivity. The cross-reactivity of Q9VF/5D-
and Q9VF/5N-specific CTLs (generated in HLA-B*0702 transgenic mice) was
tested in IFNγ-ELISpot (A) and Cr51-release
assays (B) using T1-B7 cells loaded with a single dose (1
µg/ml) (A) or a titration (B) of Q9VF/5D or
Q9VF/5N peptides. A CMV-derived HLA-B*07-restricted epitope (RPHERNGFTV,
R10TV) was used as negative control. Q9VF/5D- and Q9VF/5N-specific CTLs
displayed similar capacity to recognize cells loaded with their cognate
peptides. CTLs were also equally activated by the alternate peptides. Data
are mean values of triplicates (±SD) and representative of at least
three independent experiments.

Q9VF/5N encoding HIV strains are not recognized by Q9VF-specific CTLs. As in
Figure 2A using
T1-B7 cells infected with HIVLAI, HIVNL-AD8 or
HIVMN (X4-tropic isolate encoding Q9VF/5N). Infection rates
were equivalent (around 30% of p24+ cells). Infected cells were
then used in an IFNγ-ELISpot assay to activate Q9VF/5D- and
Q9VF/5N-specific CTLs. For each peptide, specific CTL lines were generated
in three different HLA-B*0702 transgenic mice and used in two
independent experiments. One representative experiment with one CTL line is
shown (mean values of triplicates±SD).

doi:10.1371/journal.ppat.1002049.s003

(TIF)

Acknowledgments

The authors are grateful to F. Guivel, A.Lehmann and K.Textoris-Taube for technical
assistance, D. Duffy for critical reading of the manuscript, L. Weiss, A. Samri and
G. Carcelain for help and for providing reagents. We thank Zabrina Brumme for help
in statistical analysis. We thank the ALT study group and all ALT patients for
participating in the study.